The underlying hypothesis of this research plan is that it will be possible, by appropriate manipulation of attenuated Salmonella typhi and Shigella live vectors, to develop a mucosally administered multivalent vaccine to prevent Plasmodium falciparum malaria. Proving the broad hypothesis requires that we adapt an attenuated Shigella vaccine strain to deliver eukaryotic expression plasmid-based "naked" DNA (hereafter referred to as DNA vaccine) via mucosal immunization, thereby priming the immune system to recognize P. falciparum antigens. We will then attempt to boost the immune response by administering via mucosal immunization attenuated S. typhi live vectors expressing protective antigens from the sporozoite, liver, and asexual erythrocytic stages of P. falciparum life cycle. When using such a "prime/boost" strategy in animals by priming with parenteral DNA vaccine and then boosting parenterally with a live vector expressing the relevant antigen, the immune responses and the level of protection elicited markedly exceeds that achieved when either DNA or live vector is used for both prime and boost, or when protein is used to boost following priming with DNA. The efficacy of DNA prime/live vector boost has been particularly impressive with Plasmodium antigens. To optimize the priming potential of Shigella live vectors harboring DNA vaccines, we propose to use DNA vaccines in which codon usage has been optimized for expression by mammalian (human) cells. We also propose to optimize the boosting potential of S. typhi live vectors carrying prokaryotic expression plasmids at two independent levels: 1] modifying the codon usage within falciparum open reading frames for optimum expression within S. typhi live vectors; and 2] exploiting a recently developed S. typhi plasmid maintenance system which employs partition and post-segregational killing functions, to maintain codon-optimized expression plasmids. Since we propose to study Plasmodium genes encoding CSP, LSA-1, SSP2, and MSP-1, we will examine the equivalence of priming with a mixture of four Shigella live vector strains, each carrying a plasmid encoding a distinct P. falciparum antigen, versus immunization with each individual live vector; we will also examine the equivalence of boosting with S. typhi either as a combination vaccine or as separate constructs. We hypothesize that by independently attaining partial protection against further development of each of the stages of P. falciparum, and by these effects working in concert, we will ultimately be able to prevent disease completely in some subjects and to markedly ameliorate disease severity in others.